Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
  • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
  • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
  • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
  • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
  • A61K47/6935—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
  • A61K41/0038—Radiosensitizing, i.e. administration of pharmaceutical agents that enhance the effect of radiotherapy
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
  • A61K47/547—Chelates, e.g. Gd-DOTA or Zinc-amino acid chelates; Chelate-forming compounds, e.g. DOTA or ethylenediamine being covalently linked or complexed to the pharmacologically- or therapeutically-active agent
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
  • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
  • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
  • A61K49/10—Organic compounds
  • A61K49/12—Macromolecular compounds
  • A61K49/126—Linear polymers, e.g. dextran, inulin, PEG
  • A61K49/128—Linear polymers, e.g. dextran, inulin, PEG comprising multiple complex or complex-forming groups, being either part of the linear polymeric backbone or being pending groups covalently linked to the linear polymeric backbone
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
  • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
  • A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
  • A61K49/1821—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles
  • A61K49/1824—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles
  • A61K49/1878—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles the nanoparticle having a magnetically inert core and a (super)(para)magnetic coating
  • A61K49/1881—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles the nanoparticle having a magnetically inert core and a (super)(para)magnetic coating wherein the coating consists of chelates, i.e. chelating group complexing a (super)(para)magnetic ion, bound to the surface
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/08—Solutions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
  • A61N5/00—Radiation therapy
  • A61N5/10—X-ray therapy; Gamma-ray therapy; Particle-irradiation therapy
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
  • A61N5/00—Radiation therapy
  • A61N5/10—X-ray therapy; Gamma-ray therapy; Particle-irradiation therapy
  • A61N2005/1092—Details
  • A61N2005/1098—Enhancing the effect of the particle by an injected agent or implanted device
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

• the present disclosure relates to nanoparticles and their uses in the field of medicine, in particular for the treatment of tumors by uptake of copper and/or iron.
• Tetrathiomolybdate a copper chelator which is very effective in vitro, has shown an effect in particular on the suppression of angiogenesis and tumor growth, by inducing a reduction in accessible copper [Alvarez HM, Xue Y, Robinson CD , Canalizo-Hernéndez MA, Marvin RG, Kelly RA, Mondragon A, Penner-Hahn JE, & O'Halloran T V. (2010) Tetrathiomolybdate inhibits copper trafficking proteins through metal cluster formation Science (80- ) 327(5963) 331 — 334, https://doi.org/10-1126/science.1179907].
• Zhou et al have proposed an association of a chelator within polymers which come together to form a capsule making it possible to associate another drug (resiquimod - R848).
• the chelator used is TETA.
• the particles formed are of large size (more than 400 kDa molecular weight of the RPTDH) and have a fairly limited extraction capacity, the complexing agent being quite specific for copper but of low stability.
• a copper extraction capacity has been shown for copper ion contents greater than 50 pg/ml, or 50 ppm (more than an order of magnitude of the natural concentration within our organisms).
• Wu et al proposed the use of copper-loaded nanoparticles to take advantage of the possibilities of releasing and then capturing copper to initiate an antitumor effect [Wu W, Yu L, Jiang Q, Huo M, Lin H, Wang L, Chen Y, & Shi J (2019) Enhanced Tumor-Specific Disulfiram Chemotherapy by in Situ Cu2+ Chelation-Initiated Nontoxicity-to-Toxicity Transition J Am Chem Soc 141(29) 11531-11539, https://doi.Org /10.1021 Ziacs.9b035031.
• the size of these particles based on pegylated mesoporous silica on the surface is of the order of 165 nm.
• Feng et al proposed the use of mesoporous particles of copper sulphide loaded with a drug known for its ability to complex copper (bleomycin). If this approach is interesting because of the optical properties of copper sulfide to initiate absorption in the near infrared, it must be considered that the use of copper sulfide, even charged with a complexing agent, risks causing a contribution excessive copper in some areas. For this use also, the particles are of large size (119.8 nm on average), in order to be able to encapsulate enough molecules.
• DFO Deferoxamine
• Deferoxamine which is also used for the treatment of metallic iron overload, was the first chelating agent to be used in oncology for treatment by iron sequestration. DFO has thus shown encouraging results for the treatment of leukemia and neuroblastoma in preliminary clinical trials (Wang et al., Iron and Leukemia, 2019, 38, 406).
• the use of these molecular chelates is limited by their rapid elimination, their lack of tumor targeting, their toxicity at high doses and the side effects they cause.
• Another objective of the present disclosure is to provide a compound allowing the uptake of copper and/or iron not only during its general blood circulation, but also more specifically within tumor areas.
• an objective of the present disclosure is to provide a compound which makes it possible to capture more than 10 pmol of copper and/or iron per liter or even more than 100 pmol of copper and/or iron per liter in the tumor zone, c ie locally capture 100 to 10,000 ppb of copper or iron.
• Another objective of the present disclosure is to provide a compound allowing chelation with high specificity with respect to copper and/or iron and a residence time in the tumor zones that is sufficiently long, in particular several days. , even several weeks.
• Another objective of the present disclosure is to be able to locally release ions which would replace copper and/or iron in the body, thus neutralizing its effect.
• Another objective of the present disclosure is to provide a compound whose size is sufficiently small and which allows targeting of numerous solid tumors, including metastases, and in particular bone metastases.
• Another objective of the present disclosure is to provide a compound allowing both the uptake of copper and/or iron in tumors and to provide a radiosensitizing effect for treatment by radiotherapy.
• the localized uptake of biometals should disrupt cellular repair mechanisms and amplify the effects of radiotherapy.
• nanoparticle of the following formula is proposed:
• - PS is an organic or inorganic polymer matrix
• Ch1 is a chelating group not complexed or complexed with a metal cation M1
• M1 is absent or chosen from metal cations whose complexation constant with Ch1 is lower than that of copper and/or iron, in particular at least ten times lower, for example M1 is chosen from zinc or alkaline earth metals , in particular calcium or magnesium,
• - Ch2 is a chelating group, identical to or different from the chelating group Ch1, and complexed with a metal cation M2 with a high atomic number Z greater than 40, and preferably greater than 50, characterized in that
• the n/(n+m) ratio is between 10% and 100%, preferably between 40% and 60%, and,
• the mean hydrodynamic diameter of the nanoparticle is between 1 and 50 nm, preferably between 2 and 20 nm, and more preferably between 2 and 8 nm.
• a pharmaceutical composition comprising said colloidal solution of nanoparticles and one or more pharmaceutically acceptable excipients.
• the chelating group Ch1 is chosen from those having a complexation constant with respect to copper (II) greater than 10 15 .
• the chelating group Ch1 is chosen from those having a complexation constant with copper (II) at least 10 times greater than their complexation constant with zinc and at least 10 6 times greater than their complexation constants with magnesium and calcium.
• the chelating group Ch1 is chosen from those having a complexation constant with iron (II) at least 10 times greater than their complexation constant with zinc and at least 10 6 times greater than their complexation constants with magnesium and calcium.
• At least 50% of the Ch1 is complexed with a metal cation chosen from alkaline earths.
• At least 50% of the Ch1 is complexed with zinc, calcium or magnesium.
• the chelating group Ch1 and where appropriate Ch2 is chosen from macrocyclic agents, preferably from 1,4,7-triazacyclononanetriacetic acid (NOTA), 1,4,7, 10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-l-glutaric-4,7-diacetic acid (NODAGA), and 1,4, 7,10-tetraazacyclododecane,1 -(glutaric acid)-4,7,10-triacetic acid (DOTAGA), 2,2',2”,2'”-(1,4,7,10-tetraazacyclododecane-1,4 ,7,10-tetrayl)tetraacetamide (DOTAM), and 1,4,8,11-tetraazacyclotetradecan (Cyclam), and 1,4,7,10-tetraazacyclododecane (Cyclen) and deferox
• PS is a polysiloxane matrix.
• the nanoparticle is characterized in that
• the weight ratio of silicon to the total weight of the nanoparticle is between 5% and 25%
• the total number n+m of chelating groups grafted onto the polymer is between 5 and 50 per nanoparticle, preferably between 10 and 30, and,
• the nanoparticle has an average diameter of between 2 and 8 nm.
• the nanoparticle is functionalized with a targeting agent, in particular a peptide, an immunoglobulin, a nanobody, an antibody, an aptamer or a targeting protein.
• a targeting agent in particular a peptide, an immunoglobulin, a nanobody, an antibody, an aptamer or a targeting protein.
• the metal cation M2 is chosen from radiosensitizing agents and/or contrast agents for magnetic resonance imaging, in particular gadolinium or bismuth.
• the nanoparticle is characterized in that
• PS is a polysiloxane matrix
• Ch1 and Ch2 comprise DOTAGA chelating groups of formula (I) below [Chem. 1] (I) grafted to the polysiloxane matrix by covalent bonding,
• M1 is absent and M2 is the gadolinium Gd 3+ cation
• n+m is between 5 and 50, preferably between 10 and 30, and
• the pharmaceutical composition is characterized in that it is an injectable composition for intravenous, intratumoral or intrapulmonary administration in a subject, in particular comprising an effective quantity of chelating group Ch1 for in vivo uptake of copper and/or iron in a tumor, the free chelating agent being for example at a concentration of at least 10 mM in the composition.
• the present disclosure provides a pharmaceutical composition for use in the treatment of cancer in a subject, in particular for the in vivo uptake of copper and/or iron in a tumor.
• said pharmaceutical composition may comprise an effective amount of metal cation M2, preferably gadolinium, for use as a radiosensitizing agent and the subject is treated with radiotherapy after administration of said composition.
• Figure 1 shows an HPLC-ICP/MS chromatogram of the free Gadolinium in the reaction medium as a function of the retention time Tr in minutes.
• Figure 2 shows the results of the titration of the free DOTAs of CuPRiX2o by measuring the luminescence intensity at 590 nm as a function of the amount of europium added per mg of CuPRiX2o (excitation at 395 nm).
• Figure 3 is a chromatogram of AGulX® and CuPRiX2o before and after copper complexation.
• Figure 4 shows the effect of increasing concentrations of CuPRiX2o (0, 50, 100, 500, 1000 pM free chelate equivalent to approximately 0, 150, 300, 600, 900, 1200, 1500 and 3000 pM gadolinium) on cell motility of A549 cells.
• A Quantitative analysis of wound closure as a function of time. The relative density of the wound is a measure of the density of the wound region relative to the density of the region.
• FIG. 5 shows the effect of increasing concentrations of CuPRiX2o (0, 100, 200, 300, 400 and 500 pM free chelate equivalent to approximately 0, 300, 600, 900, 1200 and 1500 pM gadolinium) on the cell motility of A549 cells.
• B Representative images of each condition, showing the original injury, as well as the injury 24 h and 48 h after. The scale bar indicates 300 ⁇ m.
• FIG. 6 shows the effect of CuPRiX20 (0 and 500 ⁇ M free chelate equivalent to about 0 and 1500 ⁇ M gadolinium) on cell motility of A549 cells. The cells were treated for 72 hours with CuPRiX2o before performing the wound.
• B Representative images of each condition, showing the original injury, as well as the injury 24 h and 48 h after. The scale bar indicates 300 pm
• FIG. 7 shows the effect of CuPRiX20 (0 and 500 pM free chelate equivalent to approximately 0 and 1500 pM gadolinium) on cell invasion of A549 cells.
• B Representative images of each condition, showing the original injury, as well as the injury 24 h and 48 h after. The scale bar indicates 300 ⁇ m.
• FIG. 9 shows the effect of the combination of CuPRiX30 with photon irradiation on the motility of A549 cells.
• Figure 10 shows the radiosensitizing effect of CuPRiX30 in A549 cells.
• Figure 11 shows the radiosensitizing effect of CuPRIX and AGulX on lines (A) A549, (B) SQ20B-CD44 and (C) 4T1.
• FIG.12 Figure 12 shows the efficacy of CuPRIXso in a mouse model of triple negative breast cancer on (A) tumor growth and (B) metastasis formation.
• A) Tumor growth is expressed as tumor volume (1/2*L*I 2 , where L is tumor length and I is tumor width) as a function of time. Results are expressed as mean ⁇ SD, *p 0.038.
• Figure 13 shows an injection/irradiation diagram
• Figure 14 shows the effectiveness of radiotherapy on tumor growth and survival
• - PS is an organic or inorganic polymer matrix
• Ch1 is a chelating group not complexed or complexed with a metal cation M1
• M1 is absent or chosen from among the metal cations whose constant of complexation with Ch1 is lower than that of copper and/or iron, in particular at least ten times lower, for example M1 is chosen from zinc or alkaline earths, in particular calcium or magnesium,
• - Ch2 is a chelating group, identical to or different from the chelating group Ch1, and complexed with a metal cation M2 with a high atomic number Z greater than 40, and preferably greater than 50, characterized in that
• the n/(n+m) ratio is between 10% and 100%, preferably between 40% and 60%, and,
• the mean hydrodynamic diameter of the nanoparticle is between 1 and 50 nm, preferably between 2 and 20 nm, and more preferably between 2 and 8 nm.
• the nanoparticles according to the present disclosure are advantageously particles with a size of the order of a nanometer.
• the nanoparticles are small enough to target tumor cells by the EPR effect via the vascular system and to be rapidly eliminated by the kidneys, after their intravenous administration.
• nanoparticles of very small diameter for example between 1 and 10 nm, preferably between 2 and 8 nm, will be used more preferably.
• the size distribution of the nanoparticles is for example measured using a commercial particle sizer, such as a Malvern Zetasizer Nano-S particle sizer based on PCS (Photon Correlation spectroscopy). This distribution is characterized by an average hydrodynamic diameter.
• diameter of the nanoparticles we thus mean the mean hydrodynamic diameter, that is to say, the harmonic mean of the diameters of the particles. A method for measuring this parameter is also described in standard ISO 13321:1996.
• the nanoparticles according to the present disclosure are nanoparticles comprising an organic or inorganic polymer PS matrix.
• the polymer of the PS matrix is chosen from biocompatible polymers such as polyethylene glycol, polyethylene oxide, polyacrylamide, biopolymers, polysaccharides or polysiloxanes, or mixtures thereof, preferably the PS polymer is a polysiloxane.
• nanoparticles comprising a matrix of polysiloxane polymers is meant in particular nanoparticles characterized by a mass percentage of silicon of at least 5%, for example between 5 and 20% of the total mass of the nanoparticle.
• polysiloxane denotes an inorganic crosslinked polymer consisting of a sequence of siloxanes.
• the structural units of the polysiloxane, which are identical or different, have the following formula: Si(OSi)nR4-n in which
• - R is an organic molecule bonded to silicon by a covalent Si-C bond
• - n is an integer between 1 and 4.
• polysiloxane includes in particular polymers resulting from the sol-gel condensation of tetraethylorthosilicate (TEOS) and aminopropyltriethoxysilane (APTES).
• TEOS tetraethylorthosilicate
• APTES aminopropyltriethoxysilane
• chelating group within the meaning of the present disclosure, is meant an organic group capable of complexing a metal cation.
• said chelating groups above are bonded directly or indirectly by covalent bond to the polysiloxane silicons of the PS matrix of the nanoparticles.
• “Indirect” bonding means the presence of a molecular “linker” or “spacer” between the nanoparticle and the chelating group, said linker or spacer being covalently bonded to one of the constituents of the nanoparticle.
• the chelating group Ch1 has in particular the function of capturing endogenous copper or endogenous iron.
• a chelating group Ch1 will advantageously be chosen from those having a complexation constant with respect to copper (II) greater than 10 15 , for example greater than 10 20 .
• a chelating group Ch1 will advantageously be chosen from those having a complexation constant with respect to iron (II) greater than 10 15 , for example greater than 10 20 .
• the chelating group Ch1 is free, or complexed (in part at least) with a metal cation M1.
• the metal cation M1 is complexed with a chelating group chosen judiciously to allow the in vivo transmetallation of the metal cation M1 with copper and/or iron.
• the chelating group Ch1 is advantageously chosen from those having a complexation constant with copper (II) or iron at least 10 times greater than their complexation constant with zinc and at least 10 6 times greater than their complexation constants with magnesium and calcium.
• the chelating group Ch1 can be obtained by grafting (covalent bond) on the nanoparticle of macrocyclic agents, preferably chosen from DOTA (acid 1,4,7,10-tetraazacyclododecane-N,N',N”,N ”'-teracetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), NODAGA (1,4,7-triazacyclononane-1-glutaric-4,7-diacetic acid), DOTAGA (2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentanedioic acid), DOTAM (1,4,7,10-tetrakis(carbamoylmethyl)-1, 4,7,10 tetraazacyclododecane) and 1,4,8,11-tetraazacyclotetradecan (Cyclam), 1,4,7,10-tetraazacycl
• the metal cation M1 complexed with the chelating group Ch1 is chosen from zinc, or alkaline earth metals, in particular magnesium or calcium.
• the chelating group Ch1 is DOTAGA of formula (I) below [Chem. 1]
• the chelating group Ch2 is complexed with a metal cation M2 with a high atomic number Z greater than 40, and preferably greater than 50.
• a nanoparticle comprises, grafted onto the polymer matrix PS, one or more Ch1 groups complexed or not with a metal cation M1, for example zinc, magnesium, calcium, or other alkaline-earth metals, and one or more Ch2 groups complexed with a metal cation M2 with number atomic Z high, greater than 40.
• the chelating group Ch2 is thus preferably chosen from among the chelating groups whose complexation constant with the metal cation M2 is greater than 10 15 , or even greater than 10 20 .
• the metal cations M2 are chosen from those allowing said nanoparticle to be used as a radiosensitizing agent.
• radiosensitizing agent means a compound making it possible to make cancerous cells more sensitive to the rays used in radiotherapy.
• the chelating group Ch2 which is identical to or different from Ch1, can also be chosen from macrocyclic agents, and preferably from DOTA (1,4,7,10-tetraazacyclododecane-N,N',N”N”' acid -teracetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), NODAGA (1,4,7-triazacyclononane-1-glutaric-4,7-diacetic acid), DOTAGA ( 2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentanedioic), DOTAM (1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10 tetraazacyclododecane) and 1,4,8,11-tetraazacyclotetradecan (Cyclam), 1,4,7,10-tetraazacyclododecane
• the metal cations M2 are chosen from heavy metals, preferably from the group consisting of: Pt, Pd, Sn, Ta, Zr, Tb, Tm, Ce, Dy, Er, Eu, La, Nd , Pr, Lu, Yb, Bi, Hf, Ho, Sm, In and Gd, or a mixture thereof.
• the metal cations M2 are Bi and/or Gd.
• the nanoparticle for the use according to the invention comprises between 3 and 100, preferably between 5 and 20 metal cations M2, in particular of Bi and/or Gd.
• the nanoparticle according to the invention allows both the uptake of copper and/or iron, by the chelating group Ch1 and/or the imaging or treatment of tumors, by the chelating group Ch2 complexed with a metal cation.
• M2 exhibiting properties of a contrast agent or of a radiosensitizing agent or of an agent for brachytherapy.
• metal cation M2 which can be used as an MRI contrast agent, mention will be made of Gd, Dy, Mn and Fe.
• metal cation M2 which can be used as a radiosensitizing agent, mention will be made of Gd, Lu, Yb and Bi, Hf and Ho, preferably gadolinium or bismuth.
• n/(n+m) is greater than or equal to 20%; in particular comprised between 20% and 100%, preferably comprised between 40% and 60%.
• n/(n+m) equals 100%. That is to say, m, representing the number of chelating agent Ch2 complexed with a metal cation M2 is equal to 0, and 100% of the chelating groups Ch are complexed with a metal cation M1 or not complexed.
• the chelating group Ch1 is identical to the chelating group Ch2 and corresponds to the DOTAGA of formula (I) below [Chem. 1] grafted onto a PS matrix of the nanoparticle, for example a polysiloxane matrix.
• the chelating group Ch1 is identical to the chelating group Ch2 and corresponds to DOTA of the following formula [Chem. 2] grafted onto a PS matrix of the nanoparticle, for example a polysiloxane matrix.
• the present disclosure relates to a [Ch1] n -PS-[Ch2] m nanoparticle wherein:
• - PS is an organic or inorganic polymer matrix
• - Ch1 is DOTA or DOTAGA not complexed or complexed with a metal cation M1,
• M1 is absent or chosen from metal cations whose complexation constant with Ch1 is lower than that of copper and/or iron, in particular at least ten times lower, for example M1 is chosen from zinc or alkaline earth metals , in particular calcium or magnesium,
• Ch2 identical to Ch1, is DOTA or DOTAGA and complexed with a metal cation M2 with a high atomic number Z greater than 40, and preferably greater than 50, preferably Gd, characterized in that
• the n/(n+m) ratio is between 10% and 100%, preferably between 40% and 60%,
• the mean hydrodynamic diameter of the nanoparticle is between 1 and 50 nm, preferably between 2 and 20 nm, and more preferably between 2 and 8 nm.
• (n+m) corresponding to the number of Ch1 and Ch2 chelating groups grafted per nanoparticle is between 3 and 100, preferably between 5 and 50, and for example between 10 and 30.
• the nanoparticles according to the present disclosure can be modified (functionalization) at the surface by hydrophilic compounds (PEG) and/or charged differently to adapt their biodistribution within the organism and/or targeting molecules to enable specific cell targeting, in particular for the targeting of specific tissues or tumor cells.
• the targeting agents are grafted to the polymer matrix and are preferably present in a proportion of between 1 and 20 targeting agents per nanoparticle and preferably between 1 and 5 targeting agents.
• the surface grafting of the targeting molecules it is possible to use a conventional coupling with reactive groups present, optionally preceded by an activation step.
• the coupling reactions are known to those skilled in the art and will be chosen according to the structure of the surface layer of the nanoparticle and the functional groups of the targeting molecule. See, for example, “Bioconjugate Techniques", GT Hermanson, Academy Press, 1996, in “Fluorescent and Luminescent Probes for Biological Activity", Second Edition, WT Mason, ed. Academic Press, 1999. Preferred coupling methods are described below.
• these targeting molecules are grafted to the amine bonds of the nanoparticles according to the variant of the ultrafine nanoparticles or AGulX as described in the following paragraph.
• the targeting molecules will be chosen according to the application envisaged.
• the nanoparticles are functionalized with a targeting agent, such as a peptide, an immunoglobulin, a nanobody, VHH fragment or "single domain", an antibody, an aptamer or any other targeting protein.
• a targeting agent such as a peptide, an immunoglobulin, a nanobody, VHH fragment or "single domain", an antibody, an aptamer or any other targeting protein.
• tumor areas typically an antibody, immunoglobulin or nanobody targeting tumor-associated antigens (“tumor-associated antigens”) or certain cancer markers known to those skilled in the art.
• the nanoparticles that can be used are nanoparticles comprising a polysiloxane PS matrix and which do not comprise a metal oxide-based core, unlike core-shell type nanoparticles comprising a core based on metal oxide and a polysiloxane coating (which are described in particular in WO2005/088314 and W02009/053644).
• the nanoparticles according to the present disclosure are nanoparticles based on polysiloxane chelated with gadolinium, of formula [Ch1] n -PS-[Ch2] m , in which
• PS is a polysiloxane matrix
• Ch1 and Ch2 are DOTAGA chelating groups of formula (I) below [Chem. 1] [0094] and grafted to the polysiloxane matrix by covalent bonding,
• M1 is absent and M2 is the gadolinium Gd 3+ cation
• n+m is between 5 and 50, preferably between 10 and 30, and
• the average hydrodynamic diameter is between 2 and 8 nm.
• these gadolinium-chelated polysiloxane-based nanoparticles are ultrafine nanoparticles obtained from AGulX nanoparticles as starting material.
• Such ultrafine AGulX nanoparticles can be obtained by a top-down synthesis method described in particular in Mignot et al Chem Eur J 2013 “A top-down synthesis route to ultrasmall multifunctional Gd-based silica nanoparticles for theranostic applications” DOI: 10.1002/chem.201203003.
• the AGulX nanoparticles which can be used as starting material for obtaining the nanoparticles according to the present disclosure, have in particular the following formula (III) [Chem. 3] in which PS is a polysiloxane matrix, and n is on average between 10 and 50, and the nanoparticles have an average hydrodynamic diameter of 4
• the AGulX nanoparticles can also be characterized by the following formula (IV) [Chem. 4] (GdSi4-7C24-30N5-8Ol5-25H40-60.5-10H20)x
• nanoparticles according to the present disclosure can be obtained by the process for preparing a colloidal solution of nanoparticles comprising chelating groups grafted onto a polymer matrix, only part of the chelating groups being complexed with a metal cation, the other part being uncomplexed, said method comprising
• - PS is an organic or inorganic polymer matrix
• - Ch2 is a chelating group complexed with a metal cation M2 with a high atomic number Z greater than 40, and preferably greater than 50, characterized in that
• Ch2 is grafted onto the polymer matrix
• n is between 5 and 100, and,
• the average hydrodynamic diameter of the NP1 nanoparticle is between 1 and 50 nm, preferably between 2 and 20 nm, and more preferably between 2 and 8 nm
• step (4) if necessary, a step of concentrating the solution of the nanoparticles obtained in step (4),
• the NP1 nanoparticles are ultrafine or AGulX nanoparticles as defined in the previous section and complexed with the gadolinium cation. Specific embodiments are given in the Examples.
• the duration of the treatment of step (2) can be between 0.5 and 8 hours, for example between 2 and 6 hours at a pH of less than 1.0.
• the nanoparticles obtained according to the above process can, if necessary, then be functionalized by other chelating groups, different from Ch2 and/or targeting agents or hydrophilic molecules.
• the nanoparticles obtained according to the above process are placed in the presence of metal cation M1, in order to obtain the complexation of at least part of the free chelating groups with the metal cation M1, of so as to obtain the nanoparticles of the following formula:
• - PS is an organic or inorganic polymer matrix
• Ch1 is a chelating group partially complexed with a metal cation M1
• M1 is chosen from metal cations whose complexation constant with Ch1 is lower than that of copper and/or iron, in particular at least ten times lower, for example M1 is chosen from zinc, calcium or magnesium or other alkaline earths,
• Ch2 is a chelating group, identical to Ch1, and complexed with a metal cation M2 with a high atomic number Z greater than 40, and preferably greater than 50, characterized in that
• the n/(n+m) ratio is between 10% and 100%, preferably between 40% and 60%, and,
• the mean hydrodynamic diameter of the nanoparticle is between 1 and 50 nm, preferably between 2 and 20 nm, and more preferably between 2 and 8 nm.
• compositions comprising the nanoparticles according to the present disclosure are administered in the form of colloidal suspensions of nanoparticles. They can be prepared as described here or according to other methods known to those skilled in the art and administered via different routes, local or systemic, depending on the treatment and the area to be treated.
• the present disclosure relates to a colloidal suspension of nanoparticles of formula [Ch1] n -PS-[Ch2] m as described in the preceding sections and the pharmaceutical compositions comprising these colloidal suspensions, where appropriate, in combination with one or more pharmaceutically acceptable excipients.
• the pharmaceutical compositions can in particular be formulated in the form of freeze-dried powders, or of aqueous solutions for intravenous injection.
• the pharmaceutical composition comprises a colloidal solution with a therapeutically effective amount of nanoparticles of formula [Ch1] n -PS-[Ch2] m as described in the previous sections, in particular nanoparticles based on chelated polysiloxane to gadolinium, and more specifically, as obtained from AGulX nanoparticles as described above.
• the present disclosure relates to a pharmaceutical composition for its use as a solution for injection, comprising, as active principle, the nanoparticles of formula [Ch1]n-PS-[Ch2] m as described in the preceding sections, in particular nanoparticles based on polysiloxane chelated with gadolinium, and more precisely, as obtained from AGulX nanoparticles as described above.
• the pharmaceutical composition is characterized in that it is an injectable composition for intravenous or intratumoral administration or an aerosol for intrapulmonary administration, in particular comprising an effective amount of chelating group Ch1 for the in vivo uptake of copper and/or iron in the tumor, the chelating agent Ch1 being for example at a concentration of at least 10 mM in the composition.
• the composition is an injectable solution comprising nanoparticles based on polysiloxane chelated with gadolinium in a concentration of between 50 and 200 mg/ml, for example between 80 and 120 mg/ml.
• the nanoparticles according to the present invention allow the uptake of endogenous copper and/or iron after their administration to a subject who needs them.
• endogenous uptake of copper we preferably mean the local uptake of a quantity of between 100 ppb (0.1 mg of copper per litre) and 10,000 ppb (10 mg of copper per litre) of endogenous copper.
• the present disclosure relates more particularly to a process for capturing endogenous copper in a subject, in particular a subject suffering from cancer.
• endogenous uptake of iron is preferably meant the local uptake of a quantity of between 100 ppb (0.1 mg of iron per litre) and 10,000 ppb (10 mg of iron per litre) of the endogenous iron. Accordingly, this disclosure is intended more particularly a method for capturing endogenous iron in a subject, in particular a subject suffering from cancer.
• the uptake of copper and/or iron can take place within the general blood circulation then afterwards within the tumors, after accumulation of the nanoparticles in tumours, in particular by passive targeting linked to the EPR effect.
• passive tumor targeting and accumulation has been well demonstrated in particular by ultrafine nanoparticles of the AGulX type.
• the disclosure therefore also relates to a method for treating tumors in a subject, said method comprising the administration to said subject, of an effective amount of a pharmaceutical composition of nanoparticles of formulas [Ch1] n -PS- [Ch2] m as described in the previous sections, in particular nanoparticles based on polysiloxane chelated with gadolinium, and more precisely, as obtained from AGulX nanoparticles as described above, and characterized in that the nanoparticles allow the treatment of the tumor partly by the uptake of endogenous copper and/or iron.
• the composition also comprises an effective amount of metal cation M2, preferably gadolinium or bismuth, for use as a radiosensitizing agent and the method comprises, after administration of the composition, a step of irradiating the subject with an effective dose for the treatment of the tumor by radiotherapy.
• metal cation M2 preferably gadolinium or bismuth
• patient or “subject” is preferably meant a mammal or a human being including, for example, a subject having a tumour.
• treatment refers to any act which aims to improve the state of health of a patient, such as therapy, prevention, prophylaxis, and the delay of a disease. In some cases, these terms refer to the amelioration or eradication of a disease or the symptoms associated with the disease. In other embodiments, these terms refer to the reduction in the spread or worsening of disease resulting from the administration of one or more therapeutic agents to a subject. suffering from such a disease.
• treatment can typically encompass a treatment for stopping the growth of a tumor, reducing the size of the tumor and/or eliminating the tumor.
• the nanoparticles are used for the treatment of solid tumors, for example brain cancer (primary and secondary, glioblastoma, etc.), hepatic cancers (primary and secondary), pelvic tumors (cancer of the cervical cancer, prostate cancer, anorectal cancer, colorectal cancer), upper aerodigestive tract cancers, lung cancer, oesophageal cancer, breast cancer, pancreatic cancer.
• solid tumors for example brain cancer (primary and secondary, glioblastoma, etc.), hepatic cancers (primary and secondary), pelvic tumors (cancer of the cervical cancer, prostate cancer, anorectal cancer, colorectal cancer), upper aerodigestive tract cancers, lung cancer, oesophageal cancer, breast cancer, pancreatic cancer.
• an effective amount of nanoparticles reference is made to the amount of nanoparticles as described above which, when administered to a patient, is sufficient to be localized in the tumor and allow treatment of the tumor by uptake of copper and / or endogenous iron, where appropriate in combination with a radiosensitizing effect and radiotherapy treatment.
• This quantity is determined and adjusted according to factors such as the age, sex and weight of the subject.
• the administration of the nanoparticles as described previously can be carried out by intratumoral, subcutaneous, intramuscular, intravenous, intradermal, intraperitoneal, oral, sublingual, rectal, vaginal, intranasal route, by inhalation or by transdermal application. Preferably, it is done intratumorally and/or intravenously.
• Irradiation methods for treating tumors after administration of nanoparticles as radiosensitizing agent are well known to those skilled in the art and have been described in particular in the following publications: WO2018/224684, WO2019/008040 and C. Verry, et al, Science Advances, 2020, 6, eaay5279; and, C. Verry, et al, NANO-RAD, a phase I study protocol”, BMJ Open, 2019, 9, e023591.
• the total dose of irradiation during radiotherapy will be adjusted according to the type of cancer, the stage and the subject to be treated.
• a dose typical total for a solid tumor is in the range of 20 to 120 Gy.
• Other factors may be taken into account such as chemotherapy treatment, co-morbidity, and/or the fact that radiotherapy takes place or after surgery.
• the total dose is usually divided.
• the radiotherapy step in the method according to the present disclosure may comprise, for example, several fractions between 2 and 6 Gy per day, for example 5 days per week, and in particular over 2 to 8 consecutive weeks, the total dose possibly being between 20 and 40 Gy, for example 30 Gy.
• nanoparticles according to the present disclosure can be administered alone, or in combination with one or more other active ingredients, and in particular other drugs such as cytotoxic or antiproliferative agents or other anti-cancer agents and in particular inhibitors immune checkpoints.
• other drugs such as cytotoxic or antiproliferative agents or other anti-cancer agents and in particular inhibitors immune checkpoints.
• combined administration is meant simultaneous or sequential administration (at different times).
• the CuPRiXx products are obtained by introducing the starting product AGulX®, supplied by the company Nh TherAguix (France), into a strongly acidic medium obtained from extra-pure 37% hydrochloric acid from Cari Roth.
• the filtration steps are carried out using a peristaltic pump and a Vivaflow 200® - 5 kDa cassette from Sartorius Stedim Biotech (France) used as under the conditions described in the instructions linked to the Vivaflow 200® product.
• the measurement of the hydrodynamic diameter as well as the titration of the isoelectric point are carried out with a Zetasizer Nano-S (633 nmHe-Ne laser) from Malvern Instruments (USA).
• this apparatus is coupled to an automatic titrator MPT-2 from Malvern Instruments (USA).
• the HPLC-UV is carried out with an Agilent 1200 with a DAD detector.
• the reverse phase column used is a C4.5 ⁇ m, 300 ⁇ , 150 x 4.6 mm from Jupiter.
• the detection is carried out by a UV detector at a wavelength of 295 nm.
• the gradient of phases A (H2O/ACN/TFA: 98.9/1/0.1) and B (H2O/ACN/TFA: 10/89.9/0.1) is as follows: 5 minutes at 95/ 5 followed by a linear gradient over 10 min which makes it possible to reach the 10/90 ratio which is maintained for 15 minutes. At the end of these 15 minutes the rate of A is returned to 95% in 1 minute and is followed by a 7 minute plateau at 95/5.
• the products used in the composition of the eluting phases are all HPLC grade certified.
• the HPLC-ICP/MS is carried out with Nexion 2000 from Perkin-Elmer (USA).
• the measurement of the free elements in the medium is carried out in isocratic mode with an elution phase of the following composition: 95% A and 5% B.
• the composition of phases A and B is identical to the HPL-UV method.
• the reverse phase column used is a C4.5 ⁇ m, 300 ⁇ , 150 ⁇ 4.6 mm from Jupiter.
• the products used in the composition of the eluting phases are all HPLC grade certified.
• the freeze-drying of the particles is carried out using an Alpha 2-4 LSC freeze-dryer from Christ (Germany) following the “primary drying” program.
• the A549 cells (ECACC 86012804) are cultured in F12-K medium (GibcoTM, Thermofischer) supplemented with 10% fetal calf serum (Dutscher) and 1% penicillin-streptomycin (GibcoTM, Thermofischer). For each experiment, the cells are rinsed twice with 1X PBS (GibcoTM, Thermofischer), incubated for 5 minutes in an incubator at 37° C., 5% CO2 with trypsin-EDTA, then taken up in complete medium.
• F12-K medium GibcoTM, Thermofischer
• 1X PBS GibcoTM, Thermofischer
• the CuPRiX2o and CuPRiXso products are taken up in sterile distilled water at a concentration of free DOTA equal to 10 mM (respectively 30 mM and 16.7 mM of gadolinium) and stored at 4°C.
• the A549 cells are seeded (50,000 cells/well) in 96-well ImageLock plates (Essen BioScience) and incubated for 16 h at 37° C. and 5% CO2 until to reach 90-100% confluence.
• the WoundMakerTM (Essen BioScience) is used to create wounds in the cell monolayer of each well. Then, each well is rinsed twice with 1X PBS.
• the A549 cells are trypsinized, centrifuged and then resuspended with depleted medium (F12-K medium without FCS). The cells are then seeded (1000 cells per well, 40 ⁇ l per well) in the IncuCyte® Clearview insert (Essen BioScience). In the corresponding wells, 20 ⁇ l of depleted medium containing or not CuPRiX20 (500 ⁇ M of free chelate) are added. Finally, 200 ⁇ l of F-12K medium at 10% FCS containing or not CuPRiX20 (500 ⁇ M of free chelate) are added to the lower compartment of the chemotaxis chamber. Images of each insert are taken every hour.
• Chemotactic migration from the upper reservoir to the lower reservoir is quantified as the confluence of cells on the underside of the membrane relative to the initial confluence of the seeded cells on the top of the membrane. The calculation is performed automatically with IncuCyte ZOOM 2015A microscopy software.
• the A549 cells are cultured in ImageLock® 96-well culture plates (Essen BioScience) at 20,000 cells per well overnight at 37° C., 5% CO2.
• the cells are treated with F-12K medium without FCS containing or not CuPRiX3o (500 pM of free chelate equivalent to 800 pM of gadolinium) for 24 h then they are irradiated at 8 Gy (X-Rad320 irradiator, 250 kV, 15 my).
• the wound is made with the 96-well WoundMakerTM (Essen BioScience).
• the cells are rinsed twice with 1X PBS to remove floating cells and 100 ⁇ l of medium containing CuPRiXso (0 and 500 ⁇ M of free chelate equivalent to approximately 0 and 800 ⁇ M of gadolinium) are added to each corresponding well.
• the cells are seeded at 40,000 cells/cm 2 , ie 1 million cells in a 25cm 2 flask (Dutscher) and incubated overnight at 37° C., 5% CO2.
• the cells are treated with medium without FCS containing or not CuPRiXso (500 ⁇ M of free chelate equivalent to 800 ⁇ M of gadolinium) for 24 h.
• the cells were then irradiated at different doses (0, 2, 3, 4, 6 and 8 Gy). After irradiation, the cells are washed with 1X PBS, trypsinized and counted. The cells are then reseeded in 25 cm 2 flasks and grow for six divisions (7 days) before being fixed and stained.
• the 4T1 line is a breast cancer cell line derived from the mammary gland of a BALB/c mouse strain.
• the cells are cultured in RPMI medium (GibcoTM, Thermofischer) supplemented with 10% FCS and 1% penicillin-streptomycin.
• the line of cancer of the upper aerodigestive tract (VADS), SQ20B, is derived from a recurrent cancer of the larynx.
• the subpopulation of interest, SQ20B/CSCs (Stemcell like cancer cells), was collected after two successive cell sorting steps (Hoechst efflux and CD44 labeling) performed by flow cytometry. This population presents a low expression of EGFR and a mesenchymal phenotype, associated with the acquisition of migratory and invasive properties.
• SQ20B-CSCs are cultured in DMEM:F12 medium (3:1, v:v) supplemented with 5% FCS, 1% PS, 0.04 mg/ml hydroctorisone and 20 ng/ml epidermal growth factor (EGF).
• DMEM:F12 medium 3:1, v:v
• FCS 5% FCS
• PS 1% PS
• hydroctorisone 0.04 mg/ml
• EGF epidermal growth factor
• mice received 3 injections of 50 ⁇ l each by intravenous route, 48 h apart, of 0.9% NaCl (control) or CuPRiX (200 mg/kg). The mice were weighed, their general state evaluated and the tumors measured 3 times per week. The mice were killed 5 days after the last injection and the metastases were quantified in the target organs (lungs, liver).
• the quantification of the metastases was carried out according to the protocol of Pulaski et al (2000). For this, after killing the mice by cervical dislocation under isoflurane anesthesia, the lungs and livers are removed. The organs are washed with HBSS, cut mechanically and then subjected to enzymatic digestion with collagenase type IV (2 mg/ml) associated with DNAse I (Roche) for the lungs and with a collagenase cocktail of type IV (2 mg/ml). I (2 mg/ml), BSA (1 mg/ml) and hyaluronidase (2 mg/ml) for the livers. This digestion takes place for 2 hours (lungs) and 40 minutes (livers) at 37°C on a rotating wheel.
• the digested organs are then filtered through 70 ⁇ m nylon filters. They are centrifuged at 1500 G for 5 minutes and washed twice with HBSS. The cell pellets are resuspended in RPMI medium at 60 pM 6-thioguanine for the study of clonogenic growth. The suspensions are put back into culture diluted to 1/6 for the lungs and pure for the livers in 10-cm petri dishes. After 8 days, the cells are fixed in 96% ethanol and stained with Giemsa (diluted 1/20 in distilled water). The clones are then counted digitally using the ColcountTM colony counter (Oxford Optronix).
• the AGulX® product was placed in an acid medium with the aim of protonating the DOTA groups and thus releasing part of the Gd 3+ ions initially complexed.
• a 200 g/L solution of AGulX® was prepared by dissolving 10 g of product in 50 ml of UltraPure water. The solution was left stirring at room temperature for 1 hour.
• a solution of 2M hydrochloric acid was prepared by adding 10 ml of 37% hydrochloric acid (Hydrochloric acid 37%, extra-pure, 2.5 L, plastic, CarIRoth) to 50 ml of UltraPure water.
• the solution was diluted by 10 with UltraPure water.
• the pH is then measured and raised to 1 ⁇ 0.2 if necessary with 1 M sodium hydroxide so as not to destroy the filtration membrane.
• the 500 ml of solution thus obtained were purified using a peristaltic pump and a Sartorius Vivaflow 200 - 5 kDa cassette in order to separate the particles from the Gd 3+ ions released in order to avoid the recomplexation of these ions.
• the initial volume of 500 ml is concentrated to 50 ml and the operation is repeated. In total, the dilution/concentration operation was repeated 4 times and the final volume is 50 ml. Following purification, the 50 ml of solution were distributed into vials each containing 2 ml of solution. The vials are placed at -80°C in order to freeze the solution and then freeze-dried to obtain our final product in the form of a brown powder.
• Example 3 Assay of the number of free DOTAGA by chelation and fluorescence of europium
• the amount of free chelates present in CuPRiX2o can be determined by europium chelation followed by a luminescence study.
• Europium indeed presents a luminescence mainly centered around 590 (5D0 -> 7F1) and 615 nm (5D0 -> 7F2). This luminescence is extinguished in the presence of water molecules.
• the principle of the assay is to add increasing amounts of europium, as long as it is chelated, the luminescence increases, then when all the chelation sites are filled the luminescence reaches a plateau as shown in Figure 2.
• the CuPRiX2o was placed in an acetate buffer at pH 5, a europium chloride salt dissolved in the acetate buffer is added. A dosage curve is then plotted by exciting at 396 nm and noting remission at 590 nm. This dosage makes it possible to go back to a quantity of chelate of 0.16 pmol per mg of CuPRiX2o. Knowing that there is a zero or negligible amount of gadolinium in the initial AGulX® product, then the initial amount of gadolinium is equal to the amount of DOTA. The initial gadolinium content of the starting material, measured by elemental analysis, was 0.81 pmol per mg of AGulX®.
• the CuPRiX2o product therefore has 20% of its DOTA groups which are free.
• the presence of free DOTA therefore indicates a potential application of CuPRiX2o as a possible chelating agent in the context of chelation therapy.
• the complexing potential of CuPRiX2o was determined by copper chelation followed by an absorbance study using HPLC-UV/Vis.
• the DOTA@Cu complex has an absorbance at 295 nm much higher than that of the DOTA@Gd complex, DOTA and copper ions in solution.
• the absorbance of CuPRiX2o will increase as the free DOTA groups complex the available copper ions, until reaching a plateau where the addition of additional copper will not cause an increase in absorbance.
• a series of samples was therefore prepared with an increasing quantity of copper chloride solution and a constant quantity of CuPRiX2o.
• the level of free DOTA can be modulated according to the reaction time of AGulX in an acid medium. After 5h, the solution was diluted by 10 with UltraPure water. The pH is then measured and raised to 1 ⁇ 0.2 if necessary with 1 M sodium hydroxide so as not to destroy the filtration membrane.
• the 500 ml of solution thus obtained are purified using a peristaltic pump and a Sartorius Vivaflow 200 - 5 kDa cassette in order to separate the particles from the Gd 3+ ions released to avoid the recomplexation of these ions
• the initial volume of 500 ml is concentrated to 50 and the operation is repeated.
• the dilution/concentration operation was repeated 4 times and the final volume is 50 ml.
• the 50 ml of solution are divided into flasks each containing 2 ml of solution.
• the vials are placed at -80°C in order to freeze the solution and then freeze-dried to obtain our final product CuPRiXso in the form of a brown powder. Once the product was obtained, a vial was removed from the batch to perform product characterizations.
• the presence of free Dota therefore indicates a potential application of CuPRiXso as a possible chelating agent in the context of chelation therapy.
• the complexing potential of CuPRiX2o was determined by copper chelation followed by an absorbance study using HPLC-UV/Vis.
• the DOTA@Cu complex has an absorbance at 295 nm much higher than that of the DOTA@Gd complex, DOTA and copper ions in solution.
• the absorbance of the product will increase as the free DOTA groups complex with the available copper ions, until it reaches a plateau where the addition of additional copper will not result in an increase in absorbance.
• Cell migration is a multi-step process which is a fundamental component of many biological and pathological processes including tumor metastasis.
• the wound test is based on the formation of a wound on a cell carpet and the study of cell motility, i.e. the ability of cells to move on a surface in response to a change in density, to close the wound. It is a direct measurement of cell motility on a solid 2D substrate.
• the objective of this example was to show the ability of CuPRiX2o to reduce cell motility.
• A549 cells were cultured in F12-K medium (Gibco) containing 8 nM CuSO4-5H2O. After having been cultured for 72 h with or without CuPRiX2o (500 ⁇ M of free chelate), the cells were trypsinized and then seeded at 40,000 cells per well in an ImageLock® 96-well culture plate (Essen BioScience). The plate was placed for overnight at 37°C, 5% CO2. The wound was then made with the IncuCyte® WoundMaker (Essen BioScience).
• the cells were rinsed twice with PBS to remove floating cells and then treated with 100 ⁇ l of medium containing increasing concentrations of CuPRiX2o (0, 50, 100, 200, 300, 400, 500 and 1000 ⁇ M of free chelate equivalent to about 0, 150, 300, 600, 900, 1200, 1500 and 3000 ⁇ M gadolinium).
• Example 8 Comparison of the effect of CuPRiX20 and CuPRiX30 on the motility of A549 cells
• the objective of this example is to show the ability of CuPRiX20 and CuPRiX30 to reduce cell motility and to compare their effects.
• A549 cells were grown in ImageLock® 96-well culture plates (Essen BioScience) at 40,000 cells per well overnight at 37°C, 5% CO2. Wound testing was performed with the 96-well IncuCyte® WoundMaker (Essen BioScience).
• Cells were rinsed 2 times with PBS to remove floating cells and were then treated with 100 ⁇ l medium containing CuPRiX20 (0, 125, 250 and 500 ⁇ M free chelate equivalent to approximately 0, 375, 750 and 1500 pM gadolinium) or CuPRiX30 (0, 125, 250 and 500 pM free chelate is equivalent to approximately 0, 200, 400 and 800 pM gadolinium.
• the images of the filling of the wounds were acquired automatically every 2 hours by the Zoom Incucyte software (Essen BioScience) in the CO2 incubator. The data was analyzed by the software and the results expressed as percentage of wound confluence.
• the objective of this example is to show the ability of CuPRiX2o to reduce cell invasion - i.e. the ability of cells to break down an extracellular matrix and to move.
• A549 cells were grown in ImageLock® 96-well culture plates at 40,000 cells per well overnight at 37°C, 5% CO2. The wound was then made with the IncuCyte® WoundMaker (Essen BioScience) then the cells were rinsed twice with PBS. 50 ⁇ l of Matrigel (corning)—diluted beforehand in F12-K medium, whether or not containing CuPRiX20—to a final concentration of 1 mg/ml, were added to each well.
• Migration by chemotaxis is the directional movement of cells in response to a stimulus.
• This test consists of a culture insert placed in a cell culture plate well. The cells are seeded in the insert - which contains a membrane with a defined pore size - with serum-free medium to starve them. The chemo-attractant medium is placed in the well below. Due to this chemical gradient, the cells capable of migrating are attracted by the chemo-attractant medium and pass through the pores.
• the objective of this example is to show the ability of CuPRiX20 to reduce cell motility by a chemotaxis assay. For this, the IncuCyte ZOOM chemotaxis module was used.
• A549 cells (1000 cells/well) were resuspended in F-12K medium containing 0% FCS and were seeded in the upper compartment of a 96-well Cell Migration Incucyte ClearView plate with 8 pm (40 ⁇ l/well).
• 20 ⁇ l of F12-K medium without FCS containing or not CuPRiX20 (500 ⁇ M of final free chelate) were added.
• 200 ⁇ l of F-12K medium at 10% FCS with or without CuPRiX20 (500 ⁇ M of free chelate) was added to the lower compartment of the chemotaxis chamber. Images of each insert were taken every hour.
• Chemotactic migration from the upper reservoir to the lower reservoir was quantified as the confluence of cells on the underside of the membrane relative to the initial confluence of cells seeded on the top of the membrane. The calculation was performed automatically with IncuCyte ZOOM 2015A microscopy software.
• Example 11 Effect of the combination of CuPRiX30 with photon irradiation on the motility of A549 cells
• the objective of this example is to show the ability of CuPRiX30 to reduce cell motility after photon irradiation.
• A549 cells were grown in ImageLock® 96-well culture plates at 20,000 cells per well overnight at 37°C, 5% CO2. The cells were incubated with F-12K medium without FCS containing or not CuPRiX30 (500 pM free chelate equivalent to 800 pM gadolinium) for 24 h. The cells were then irradiated at 8 Gy (X-Rad320 irradiator, 250 kV) and then wounding was performed. The cells were rinsed twice with PBS to remove floating cells and 100 ⁇ l of medium containing CuPRiX30 (0 and 500 ⁇ M free chelate equivalent to about 0 and 800 ⁇ M gadolinium) was added to each corresponding well.
• the images of the filling of the wounds were acquired automatically every 2 hours by the Zoom Incucyte software in the CO2 incubator.
• the data was analyzed by the software and the results expressed as percentage of wound confluence.
• the results obtained showed superior efficacy (additive effect) of the CuPRiX30/irradiation combination in limiting motility compared to treatment with irradiation alone or with CuPRiX30 alone ( Figure 9).
• Example 12 Effect of the combination of CuPRiX30 with photon irradiation on cell survival of A549 cells
• the A549 cells were seeded at 40,000 cells/cm2, ie 1 million cells in a T25cm2 flask and incubated overnight at 37° C., 5% CO2.
• the cells were treated with F-12K medium without FCS containing or not CuPRiX30 (500 pM free chelate equivalent to 800 pM gadolinium) for 24 h.
• the cells were then irradiated at different doses (0, 2, 3, 4, 6 and 8 Gy). After irradiation, cells were washed with PBS, trypsinized and counted. Cells were then reseeded into 25 cm2 flasks and allowed to grow for six divisions (7 days) before being fixed and stained.
• the results obtained show a decrease in cell survival after irradiation in the presence of CuPRiXso (FIG. 10).
• Example 13 Effect of the combination of CuPRiXso with photon irradiation on cell survival of A549, SQ20B-CD44 + and 4T1 cells.
• the cells were seeded at 40,000 cells/cm 2 , i.e. 1 million cells in a 25 cm 2 flask and incubated on overnight at 37°C, 5% CO2.
• the medium is then removed and replaced with medium without FCS alone, containing CuPRiXso (500 ⁇ M of free chelate equivalent to 800 ⁇ M of gadolinium) or AGulX® (800 ⁇ M of gadolinium) for 24 h.
• the cells are then irradiated at different doses (0, 2, 3, 4 and 6 Gy). After irradiation, the cells are washed with PBS, trypsinized and counted.
• SF e - (- aD + P D2 )
• SF the surviving fraction
• a and p represent the probabilities of lethal and sublethal damage, respectively.
• the results obtained show a reduction in cell survival after irradiation after treatment with AGulX and CuPRiXso.
• the efficacy of CuPRiXso seems equal to that of AGulX, while it is superior to the AGulX in the case of the 4T 1 line (FIG. 11).
• Example 14 Efficacy of CuPRiXso on tumor growth and the formation of metastases in a mouse model of metastatic breast cancer
• the objective of this example is to show the capacity of CuPRiXso to slow down tumor growth and to reduce the formation of metastases.
• 10 female BALB/c mice aged 8 weeks received a subcutaneous injection of 50,000 4T1 cells (triple negative breast cancer cells, 50:50, PBS:matrigel).
• Two new injections were administered 48 h (D12) and 96 h (D14) after the first.
• mice Nineteen days after the inoculation of the tumours, ie 5 days after the last injection, the mice were killed by cervical dislocation under isoflurane anesthesia and the organs which could present metastases (lungs and liver) were removed. For the quantification of metastases, the lungs and livers were mechanically dissociated and then underwent enzymatic digestion before being filtered. The cell suspensions are then cultured pure (livers) or diluted (lungs) in culture medium containing 60 ⁇ M of 6-thioguanine (selection agent for 4T1 cells), and incubated at 37° C. in a CO2 incubator. Eight days later, the cells are fixed, stained and the colonies are counted automatically, the results are listed in Table 1.
• Example 15 Efficacy of the CuPRiX30 and radiotherapy combination on tumor growth and survival in a mouse model of metastatic breast cancer
• the objective of this example is to show the ability of CuPRiXso to increase the effectiveness of radiotherapy on tumor growth and survival.
• 42 female BALB/c mice aged 8 weeks received a subcutaneous injection of 50,000 4T1 cells (triple negative breast cancer cells, 50:50, PBS:matrigel).
• mice received a total of 3 injections of 50 ⁇ l of CuPRiX (200 mg/ml) or NaCl (0.9%) spaced 48 hours apart, associated with fractionated radiotherapy of 5 x 2 Gy (2 Gy per day for 5 days) (Figure 13).
• the irradiations took place 1 hour after administration of the treatment, under isoflurane anesthesia.
• mice were weighed and the tumors measured 6 times a week. At the end of the therapeutic sequence, the mice having reached one of the following endpoints: loss of 15% of its weight; tumor volume > 1000 mm3; persistent ulceration; observation of signs of distress (prostration, rough hair, hunched back) for 2 consecutive days; were put to death.
• the evolution of the tumor volume was calculated as follows: (Tumor volume at time t)/(Tumor volume at reference time) with the reference time corresponding to the first day of treatment, i.e. D10.
• the results obtained show a greater reduction in tumor growth after the combination of CuPRiX + RT treatment compared to treatment with RT alone or CuPRiX alone.
• the combination of CuPRiX with RT also prolonged animal survival compared to RT alone ( Figure 14).
• the present technical solutions can find application in particular in the field of medicine, in particular for the treatment of tumours.

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